【摘要】  目的 构建针对人caspase8 基因mRNA的siRNA表达载体，并观察其对转染细胞中caspase8的抑制作用。方法 化学合成针对caspase8发夹状的RNA寡核苷酸，将其退火形成双链，连接入经HindⅢ和BglⅡ双酶切后的pSUPER真核表达载体。对重组质粒进行酶切分析和测序鉴定。通过脂质体介导，把重组质粒瞬时转染入HeLa细胞，RTPCR测其对mRNA和蛋白表达的干涉效果。结果 成功地构建了针对人caspase8基因的RNA干涉真核表达载体pSUPERC1和pSUPERC2，并在人HeLa细胞中有效发挥了对caspase8基因的干涉作用，而且pSUPERC1对于caspase8基因的抑制作用要优于pSUPERC2。结论 成功地构建了针对人caspase8基因的siRNA载体，转染HeLa细胞后可抑制caspase8基因的表达。

【关键词】  caspase8；RNA干涉；真核表达载体；HeLa细胞

    Abstract:Objective  To construct the eukaryotic expression vector for RNA interferencing human caspase8 gene and detect its interference effect HeLa cell line. Methods  Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors：pSUPERC1 and pSUPERC2．The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing Then HeLa cells were transfected with pSUPERC1 or pSUPERC2， the interference effect was detected by RTPCR. Results  Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSUPER vector respectively．The results of RTPCR indicated that both vectors could knock down the transcription and expression of caspase gene，and that pSUPERC1 had be……